Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)

This document specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when diluted with hard water or - in the case of ready-to-use products - with water. Products can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance.
The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. This document applies to products that are used for equipment disinfection by immersion, surface disinfection by wiping, spraying, flooding or other means and teat disinfection in the veterinary area - e.g. in the breeding, husbandry, production, veterinary care facilities, transport and disposal of all animals except when in the food chain following death and entry into processing industry. This document also applies to products used for teat disinfection in these veterinary areas.
This method is not applicable to evaluate the activity of hand hygiene products. For these products reference is made to EN 14885, which specifies in detail the relationship of the various tests to one another and to "use recommendations".
NOTE   This method corresponds to a phase 2 step 1 test.

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die bakterizide Wirkung von chemischen Desinfektionsmitteln und antiseptischen Produkten fest, die bei Verdünnung in Wasser standardisierter Härte oder - im Falle gebrauchsfertiger Produkte - in Wasser als homogenes und physikalisch stabiles Präparat vorliegen. Die Produkte können nur bei einer Konzentration von höchstens 80 % geprüft werden, da durch die Zugabe der Prüforganismen und der Belastungssubstanz immer eine bestimmte Verdünnung auftritt.
Das beschriebene Verfahren soll der Bestimmung der Wirkung handelsüblicher Zubereitungen oder von Wirkstoffen unter den Bedingungen dienen, unter denen sie in der Praxis angewendet werden. Dieses Dokument gilt für Produkte, die für die Instrumentendesinfektion durch Eintauchen und bei der Oberflächendesinfektion durch Abwischen, Besprühen, Überfluten oder auf sonstige Weise sowie bei der Zitzendesinfektion im Veterinärbereich verwendet werden - z. B. bei der Aufzucht, Haltung, Produktion, in veterinärischen Pflegeeinrichtungen, beim Transport von Tieren sowie bei der Tierkörperbeseitigung aller Tiere mit Ausnahme der Tiere, die nach der Tötung direkt als Nahrungsmittel verwendet oder der weiterverarbeitenden Industrie zugeführt werden. Dieses Dokument gilt auch für Produkte, die bei der Zitzendesinfektion in diesen Veterinärbereichen verwendet werden.
Dieses Verfahren kann nicht angewendet werden, um die Wirkung von Produkten der Handhygiene zu überprüfen. Für diese Produkte wird auf EN 14885 verwiesen, die im Einzelnen den Zusammenhang zwischen den verschiedenen Prüfungen sowie zu den "Anwendungsempfehlungen" festlegt.
ANMERKUNG   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.

Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l’évaluation de l’activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d’essai et exigences (phase 2, étape 1)

Le présent document spécifie une méthode d’essai et les exigences minimales relatives à l’activité bactéricide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans de l’eau dure ou, dans le cas de produits prêts à l’emploi, dans l’eau. Les produits ne peuvent être soumis à l’essai qu’à la concentration de 80 % ou à des concentrations inférieures, car l’ajout des microorganismes d’essai et de la substance interférente s’accompagne forcément d’une dilution.
La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives dans les conditions d’utilisation. Le présent document s’applique aux produits utilisés dans la désinfection des équipements par immersion, la désinfection de surfaces par essuyage, pulvérisation, inondation ou d’autres moyens, la désinfection des trayons dans le domaine vétérinaire, par exemple dans la reproduction, l’élevage, la production, les services vétérinaires, le transport et l’abattage de tous les animaux, à l’exception de la chaîne alimentaire qui suit l’abattage et l’entrée dans l’industrie de transformation. Le présent document s’applique également aux produits utilisés dans la désinfection des trayons dans ces domaines vétérinaires.
Cette méthode ne s’applique pas à l’évaluation de l’activité des produits d’hygiène des mains. Pour ces produits, se référer à l’EN 14885, qui spécifie de façon détaillée les relations des différents essais entre eux et avec les « recommandations d’emploi ».
NOTE   Cette méthode correspond à un essai de phase 2, étape 1.

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 1)

Ta evropski standard določa preskusno metodo in minimalne zahteve za baktericidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma z vodo pri izdelkih, ki so pripravljeni za uporabo. Izdelke je mogoče preskušati samo pri 80-odstotni ali nižji koncentraciji, ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje.
Ta evropski standard se uporablja za izdelke v veterini, tj. pri vzreji, živinoreji, prevozu in odstranjevanju vseh živali, razen če so v prehrambeni verigi po smrti in so del predelovalne industrije.
Standard EN 14885 podrobno določa razmerje med različnimi preizkusi in priporočili za uporabo.
OPOMBA 1:   Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi pod pogoji, v katerih se uporabljajo.
OPOMBA 2:   Ta metoda ustreza preskusu stopnje 1 faze 2.

General Information

Status
Published
Publication Date
17-Sep-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
18-Sep-2019
Completion Date
18-Sep-2019

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SLOVENSKI STANDARD
SIST EN 1656:2019
01-november-2019
Nadomešča:
SIST EN 1656:2010
SIST EN 1656:2010/AC:2010
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za

vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v veterini -

Preskusna metoda in zahteve (faza 2, stopnja 1)

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation

of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary

area - Test method and requirements (phase 2, step 1)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur

Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika

für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour

l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques

utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase 2, étape 1)

Ta slovenski standard je istoveten z: EN 1656:2019
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
SIST EN 1656:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 1656:2019
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SIST EN 1656:2019
EN 1656
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2019
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes EN 1656:2009
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of bactericidal activity of
chemical disinfectants and antiseptics used in the
veterinary area - Test method and requirements (phase 2,
step 1)

Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -

quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der

bactéricide des antiseptiques et des désinfectants bakteriziden Wirkung chemischer Desinfektionsmittel

chimiques utilisés dans le domaine vétérinaire - und Antiseptika für den Veterinärbereich -

Méthode d'essai et exigences (phase 2, étape 1) Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

This European Standard was approved by CEN on 28 July 2019.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 1656:2019 E

worldwide for CEN national Members.
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SIST EN 1656:2019
EN 1656:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .......................................................................................................................................................................... 6

2 Normative references .......................................................................................................................................... 6

3 Terms and definitions ......................................................................................................................................... 6

4 Requirements ......................................................................................................................................................... 6

5 Test method ............................................................................................................................................................ 8

5.1 Principle .......................................................................................................................................................... 8

5.2 Materials and reagents ............................................................................................................................... 8

5.2.1 Test organisms ..................................................................................................................................... 8

5.2.2 Culture media and reagents ............................................................................................................ 9

5.3 Apparatus and glassware ....................................................................................................................... 11

5.3.1 General ................................................................................................................................................. 11

5.3.2 Usual microbiological laboratory equipment and, in particular, the following: ....... 12

5.4 Preparation of test organism suspensions and product test solutions ................................. 13

5.4.1 Test organism suspensions (test and validation suspension) ......................................... 13

5.4.2 Product test solutions ..................................................................................................................... 14

5.5 Procedure for assessing the bactericidal activity of the product ............................................ 15

5.5.1 General ................................................................................................................................................. 15

5.5.2 Dilution-neutralization method ................................................................................................. 16

5.5.3 Membrane filtration method........................................................................................................ 18

5.6 Experimental data and calculation ..................................................................................................... 20

5.6.1 Explanation of terms and abbreviations .................................................................................. 20

5.6.2 Calculation .......................................................................................................................................... 20

5.7 Verification of methodology .................................................................................................................. 23

5.7.1 General ................................................................................................................................................. 23

5.7.2 Control of weighted mean counts ............................................................................................... 23

5.7.3 Basic limits .......................................................................................................................................... 24

5.8 Expression of results and precision ................................................................................................... 24

5.8.1 Reduction ............................................................................................................................................ 24

5.8.2 Control of active and non-active product test solution (5.4.2) ........................................ 24

5.8.3 Limiting test organism and bactericidal concentration ..................................................... 24

5.8.4 Precision, replicates ........................................................................................................................ 24

5.9 Interpretation of results - conclusion ................................................................................................ 25

5.9.1 General ................................................................................................................................................. 25

5.9.2 Bactericidal activity for surface disinfection products ...................................................... 25

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SIST EN 1656:2019
EN 1656:2019 (E)

5.9.3 Bactericidal activity for teat disinfection products ............................................................. 25

5.10 Test report ................................................................................................................................................... 25

Annex A (informative) Referenced strains in national collections ........................................................... 27

Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of chemical

disinfectants and antiseptics and rinsing liquids ........................................................................................... 29

Annex C (informative) Dilution-neutralization method ................................................................................ 31

Annex D (informative) Example of a typical test report ................................................................................ 36

Bibliography ................................................................................................................................................................. 40

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SIST EN 1656:2019
EN 1656:2019 (E)
European foreword

This document (EN 1656:2019) has been prepared by Technical Committee CEN/TC 216 “Chemical

disinfectants and antiseptics”, the secretariat of which is held by AFNOR.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by March 2020, and conflicting national standards shall be

withdrawn at the latest by March 2020.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This European Standard supersedes EN 1656:2009 and was revised to harmonize the structure and

wording with other quantitative suspension tests of CEN/TC 216 (existing or in preparation).

Results obtained using the previous version of this standard are still valid.

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,

Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,

Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North

Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United

Kingdom.
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SIST EN 1656:2019
EN 1656:2019 (E)
Introduction

This document specifies a suspension test for establishing whether a chemical disinfectant or antiseptic

has a bactericidal activity in the fields described in the scope.

This laboratory test takes into account practical conditions of application of the product, including

contact time, temperature, test organisms and interfering substances, i.e. conditions which may influence

its action in practical situations.

The conditions are intended to cover general purposes and to allow reference between laboratories and

product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test

corresponds to defined experimental conditions. However, for some applications the recommendations

of use of a product may differ and therefore additional test conditions need to be used.

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SIST EN 1656:2019
EN 1656:2019 (E)
1 Scope

This document specifies a test method and the minimum requirements for bactericidal activity of

chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation

when diluted with hard water or - in the case of ready-to-use products - with water. Products can only be

tested at a concentration of 80 % or less, as some dilution is always produced by adding the test

organisms and interfering substance.

The method described is intended to determine the activity of commercial formulations or active

substances under the conditions in which they are used. This document applies to products that are used

for equipment disinfection by immersion, surface disinfection by wiping, spraying, flooding or other

means and teat disinfection in the veterinary area – e.g. in the breeding, husbandry, production,

veterinary care facilities, transport and disposal of all animals except when in the food chain following

death and entry into processing industry. This document also applies to products used for teat

disinfection in these veterinary areas.

This method is not applicable to evaluate the activity of hand hygiene products. For these products

reference is made to EN 14885, which specifies in detail the relationship of the various tests to one

another and to “use recommendations”.
NOTE This method corresponds to a phase 2 step 1 test.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the

determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal

(including bacteriophages) activity

EN 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical

disinfectants and antiseptics
3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 14885 apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
4 Requirements

The product shall demonstrate at least a 5 decimal log (lg) reduction when diluted with hard water

(5.2.2.7) or – in the case of ready-to-use products – with water (5.2.2.2) and tested in accordance with

Table 1 and Clause 5 under simulated low level soiling (3 g/l bovine albumin) or high level soiling (10 g/l

yeast extract and 10 g/l bovine albumin) or 10 g/l skimmed milk for post-milking teat disinfectants or

3 g/l bovine albumin for pre-milking teat disinfectants or in additional test conditions.

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SIST EN 1656:2019
EN 1656:2019 (E)
Table 1 — Test conditions
Bactericidal
Bactericidal activity for teat
Test conditions activity on
disinfectants
surfaces
Minimum spectrum of Enterococcus hirae Escherichia coli
test organisms
Proteus hauseri ) Staphylococcus aureus
Pseudomonas Streptococcus uberis
aeruginosa
Staphylococcus
aureus
additional any relevant test any relevant test organism
organism
Test temperature at intervals of 5 °C
minimum 5 °C ± 1 °C 20 °C ± 1 °C
maximum 40 °C ± 1 °C 30 °C ± 1 °C
Contact time at intervals of 30 s from 30 s to 5 min and at intervals
of 5 min from 5 min to 120 min
minimum 1 min ± 5 s
1 min ± 5 s for post-milking teat
disinfectants
30 s ± 5 s for pre-milking teat
disinfectants
maximum 120 min ± 10 s
30 min ± 10 s for post-milking teat
disinfectants
3 min ± 10 s for pre-milking teat
disinfectants
Interfering substance Interfering substance
low level soiling 3,0 g/l bovine albumin Post milking: 10,0 g/l of milk
powder
high level soiling 10 g/l yeast extract
plus 10 g/l bovine Pre-milking: 3,0 g/l bovine
albumin albumin
additional any relevant any relevant substance
substance
Was known as Proteus vulgaris.
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SIST EN 1656:2019
EN 1656:2019 (E)

Any additional specific bactericidal activity shall be determined in accordance with 5.2.1 and 5.5.1.1 in

order to take into account intended specific use conditions.
5 Test method
5.1 Principle

5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready-to-use

products) is added to a test suspension of bacteria in a solution of an interfering substance. The mixture

is maintained at the test temperature θ for the test contact time t. At the end of this contact time, an

aliquot is taken, and the bactericidal and/or the bacteriostatic activity in this portion is immediately

neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a

suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving bacteria in

each sample are determined and the reduction is calculated.

5.1.2 For general disinfectant products, the test is performed using Enterococcus hirae, Proteus hauseri,

Pseudomonas aeruginosa and Staphylococcus aureus as test organisms. For teat disinfectants the test is

performed using Escherichia coli, Staphylococcus aureus and Streptococcus uberis as test organisms.

5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms

can be used.
5.2 Materials and reagents
5.2.1 Test organisms

The bactericidal activity shall be evaluated using the following strains as test organisms: )

a) General disinfection products
- Enterococcus hirae ATCC 10541
- Proteus hauseri ATCC 13315
- Pseudomonas aeruginosa ATCC 15442
- Staphylococcus aureus ATCC 6538
b) Teat disinfectants
- Escherichia coli ATCC 10536
- Staphylococcus aureus ATCC 6538
- Streptococcus uberis ATCC 19436
NOTE Refer to Annex A for strain references in some other culture collections.

The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3). The

same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its

control and validation.

The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC) This

information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product

named.
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SIST EN 1656:2019
EN 1656:2019 (E)

If additional test organisms are used, they shall be incubated under optimum growth conditions

(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms selected

do not correspond to the specified strains, their suitability for supplying the required inocula shall be

verified. If these additional test organisms are not classified at a reference centre, their identification

characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture

collection under a reference for five years.
5.2.2 Culture media and reagents
5.2.2.1 General

All weights of chemical substances given in this standard refer to the anhydrous salts. Hydrated forms

may be used as an alternative, but the weights required shall be adjusted to allow for consequent

molecular weight differences.

The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be

free from substances that are toxic or inhibitory to the test organisms.

To improve reproducibility, it is recommended that commercially available dehydrated material is used

for the preparation of culture media. The manufacturer's instructions relating to the preparation of these

products should be rigorously followed.

For each culture medium and reagent, a shelf life should be fixed (see ISO/IEC 17025:2017).

5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water.
Sterilize in the autoclave [5.3.2.1 a)].
Refer to 5.2.2.7 for the procedure to prepare hard water.

NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently

sterilized.

NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference

[1]) can be used.
5.2.2.3 Tryptone Soya Agar (TSA)
Tryptone soya agar, consisting of:
— Tryptone, pancreatic digest of casein 15,0 g;
— Soya peptone, papaic digest of soybean meal 5,0 g;
— Sodium chloride (NaCl) 5,0 g;
— Agar 15,0 g;
— Water (5.2.2.2) to 1 000,0 ml.

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the medium shall be equivalent to

7,2 ± 0,2 when measured at (20 ± 1) °C.

In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add

neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used.

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EN 1656:2019 (E)
5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:
— Tryptone, pancreatic digest of casein 1,0 g;
— Sodium chloride (NaCl) 8,5 g;
— Water (5.2.2.2) to 1 000,0 ml.
— Sterilize in the autoclave [5.3.2.1 a)].

— After sterilization, the pH of the diluent shall be equivalent to 7,0 ± 0,2 when measured at (20 ± 1) °C.

5.2.2.5 Neutralizer

The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and

5.5.2. It shall be sterile.

Information on neutralizers that have been found to be suitable for some categories of products is given

in Annex B.
5.2.2.6 Rinsing liquid (for membrane filtration)

The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and

5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter

membrane under the test conditions described in 5.5.3.

Information on rinsing liquids that have been found to be suitable for some categories of products is given

in Annex B.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 000 ml of hard water, the procedure is as follows:

— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride

(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the

autoclave [5.3.2.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to

1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8)

for no longer than one month;

— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to

1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) for

no longer than one week;

— place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml

(5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The

pH of the hard water shall be 7,0 ± 0,2 (5.3.2.4) when measured at (20 ± 1) °C. If necessary, adjust

the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or

approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).

The hard water shall be freshly prepared under aseptic conditions and used within 12 h.

NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water

produces a different final water hardness in each test tube. In any case the final hardness is lower than 300 mg/l of

calcium carbonate (CaCO ) in the test tube.
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SIST EN 1656:2019
EN 1656:2019 (E)
5.2.2.8 Interfering substance
5.2.2.8.1 General

The interfering substance shall be chosen according to the conditions of use laid down for the product.

The interfering substance shall be sterile and prepared at 10 times its final concentration in the test. The

ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral

substances, protein, carbohydrates, lipids and detergents) shall be defined.

NOTE The term “interfering substance” is used even if it contains more than one substance.

5.2.2.8.2 Low-level soiling and Pre-milking teat disinfection (bovine albumin solution)

Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water

(5.2.2.2).

Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.

The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l.

5.2.2.8.3 High-level soiling (mixture of bovine albumin solution with yeast extract)

Dissolve 50,0 g yeast extract powder in 150 ml of water (5.2.2.2) in a 250 ml volumetric flask (5.3.2.12)

and allow foam to collapse. Make up to the mark with water (5.2.2.2). Transfer to a clean dry bottle and

sterilize in an autoclave [5.3.2.1 a)]. Allow to cool to (20 ± 1) °C.

Pipette 25 ml of this solution into a 50 ml volumetric flask (5.3.2.12) and add 10 ml of water (5.2.2.2).

Dissolve 5,0 g of bovine albumin fraction V (suitable for microbiological purposes) in the solution with

shaking and allow foam to collapse. Make up to the mark with water (5.2.2.2), sterilize by membrane

filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.

The final concentration in the test procedure (5.5) is 10,0 g/l yeast extract and 10,0 g/l bovine albumin.

5.2.2.8.4 Milk for post milking teat disinfection

Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder

per litre of water (5.2.2.2), shall be prepared as follows:

— prepare a solution of 100 g milk-powder in 1 000 ml water (5.2.2.2). Heat for 30 min at (105 ) °C

or 5 min at (121 ) °C.
The final concentration in the test procedure (5.5) is 10,0 g/l milk powder.
5.3 Apparatus and glassware
5.3.1 General

Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and

reagents or the sample, except those which are supplied sterile, by one of the following methods:

a) by moist heat, in the autoclave [5.3.2.1 a)];
b) by dry heat, in the hot air oven [5.3.2.1 b)].
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SIST EN 1656:2019
EN 1656:2019 (E)

5.3.2 Usual microbiological laboratory equipment and, in particular, the following:

5.3.2.1 Apparatus for sterilization:

a) for moist heat sterilization, an autoclave capable of being maintained at (121 ) °C for a minimum

holding time of 15 min;

b) for dry heat sterilization, a hot air oven capable of being maintained at (180 ) °C for a minimum

+5 +5

holding time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a

0 0
minimum holding time of 2 h.

5.3.2.2 Water baths, capable of being controlled between 5 and 40 °C ± 1 °C as required by the test

conditions, and at 45 °C ± 1 °C (to maintain melted TSA in case of pour plate technique).

5.3.2.3 Incubator, capable of being controlled either at 36 °C ± 1 °C or 37 °C ± 1 °C.

5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.

A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media

(5.2.2.3).
5.3.2.5 Stopwatch.
5.3.2.6 Shaker
a) Electromechanical agitator e.g. Vortex® mixer );
b) mechanical shaker.

5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances

to be filtered.

The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of

diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7), bovine albumin

(5.2.2.8.2 and 5.2.2.8.3) and if the membrane filtration method (5.5.3) is used.

The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution

of the microorganisms over the membrane and to prevent overlong filtration, the device shall be set so

as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes, of nominal c
...

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